Dear researchers
I am looking for a procedure how to construct a vector for promoter deletion analysis. Especially, I mean how to generate deletion fragments of a gene promoter for further cloning into a vector. Is it better to generate these fragments by digestion of the promoter sequence with using restriction enzymes. Maybe a better way will be to generate such promoter fragments by PCR amplification. How to determine then the length of the amplified promoter fragments for further cloning. Are there any rules or if I can set them arbitrarily. I will be grateful for any tips.
Hi,
I did it the way that I first inserted the full lenght promoter into a suitable Luciferase reporter vertor. Then I started to generate binding motif mutants using overlap exntension PCR. With this method you can insert or Delete long stretches
sven
In the old days one would clone the promoter in pBluescript and do ExoIII/Mung Bean to generate deletions. Right now, the quickest way would be to ask a DNA synthesis company to produce the series of progressively shorter promoters (and possibly, to provide them already cloned in the reporter vector of your choice). Hope it helps!
Recently i done this experiment, at first i have cloned the longest fragment (2000bp) into luciferase reporter vector (pGL3), then using this longer fragment as a template, i amplified the shorter fragments through PCR with enzymes site added into the primers.
Hopefully, these papers may be helpful
Thank you for many answears. I try to find rules indicating how to divide the promoter for individual deletion sequences. Is the length for all of these sequences is somehow selected or determined arbitraily. Will I find any universal protocol for this method because in various publications these fragments are determined by cutting the promoter sequence by some restriction enzymes, while in others publications they are amplified by PCR. How to choose such restriction enzymes or how long the region should be amplified for each of the deletion promoter fragments. It is hard to find any information on these matters.
You should select promoter fragments based on TFs binding sites. Then design forward primers for all selected fragments while reverse primer will be the same for all fragments. First fragment will be started most upstream most distal from the transcription start site then delete 250-300bp from the first fragment, again design 2nd forward primer and so on, in this way come closer to the transcription start site.
for amplification and clonning add restriction enzyme site to all primers and then after PCR amplification cut the fragments accordingly.
Thanks Rajwali Khan. I understand that restriction enzyme site which should be added to all primers can not be on the vector sequence used for clonning.
Dear Rajwali Khan. I understand that restriction enzyme site which should be added to all of the primers can not exist in the vector sequence used for clonning.
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